Blood -- Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT

Blood, Vol. 113, Issue 24, 6128-6137, June 11, 2009

Cytoskeletal protein 4.1R negatively regulates T-cell activation by inhibiting the phosphorylation of LAT
Blood Kang et al. 113: 6128

Supplemental materials for: Kang et al

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Table S1. Phenotyping of major lymphoid organs (PDF, 17.3 KB)
Figure S1. 4.1R is recruited to the interface between T-cell– and antigen-presenting cell (JPG, 40.9 KB) -
(A) Primary T-cell blasts from 5c.c7 transgenic mice were stained with anti-4.1R exon 13 polyclonal antibody, followed by anti-rabbit Alexa-488–conjugated secondary antibody, and analyzed by confocal microscopy. Note that protein 4.1R is evenly distributed around the cell membrane. (B) Primary T-cell blasts stimulated by peptide-loaded CH27 cells were stained as described above. Note that protein 4.1R becomes enriched at the interface between the T cell and the APC cell. Scale Bar, 2 µm. Images shown in both panel A and panel B are collage of images from four different fields.
Figure S2. Expression of 4.1R in thymocytes and enhanced signaling in 4.1R^−∕− thymocytes (JPG, 36.5 KB) -
(A) themocytes (1 × 10⁶ cells) from 4.1R^+∕+ or 4.1R^−∕− mice were subjected to immunoblot analysis with polyclonal rabbit antibodies against 4.1R exon 13. The position of ~ 135 kDa and ~ 80 kDa 4.1R is indicated. (B) 4.1R^+∕+ or 4.1R^−∕− thymocytes were stimulated with anti-CD3ε (5 µg/ml) for varying time periods, and indicated proteins were immunoblotted for the level of expression and for the extent of phosphorylation. (C)The enhanced phosphorylation of both LAT and ERK in 4.1R^−∕− thymocytes is significant.
Figure S3. SDS-PADE of recombinant proteins used in the present study (JPG, 21 KB) -
1 µg of GST or His fusion protein was analyzed by 10% SDS-PAGE and stained with GelCode Blue. This picture is a collage from two gels.
Figure S4. Interaction of cytoplasmic domain of LAT with Lobe C of 4.1R as assessed by surface plasmon resonance assay (JPG, 49 KB) -
GST-tagged Lobe C of 4.1R was immobilized onto CM5 sensor chip. His-tagged cytoplasmic domain of LAT at different concentrations was injected at 20 µl/min over the surface in a BIAcore 3000 instrument. The figure shows dose-response curves of LAT binding to the immobilized Lobe C.